Method of preserving early embryos of experimental animals by vitrification

Abstract

This invention provides a solution for preserving mammalian early embryos or ES cells by vitrification, which comprises, as a base material, a phosphate buffer that exclusively contains 10% to 15% (v/v) propylene glycol as polyhydric alcohol or a phosphate buffer that exclusively contains 10% to 15% (v/v) propylene glycol and 25% to 35% (v/v) ethylene glycol as polyhydric alcohols and further contains 15% to 25% (v/v) Percoll® and 0.2 M to 0.5 M sucrose. This invention also provides a method for preserving mammalian early embryos or ES cells by vitrification using such solution.

1 . A solution for preserving mammalian early embryos or ES cells by vitrification comprising, as a base material, a phosphate buffer that exclusively contains 10% to 15% (v/v) propylene glycol as polyhydric alcohol.  
   
   
       2 . A solution for preserving mammalian early embryos or ES cells by vitrification comprising, as a base material, a phosphate buffer that exclusively contains 10% to 15% (v/v) propylene glycol and 25% to 35% (v/v) ethylene glycol as polyhydric alcohols and further contains 15% to 25% (v/v) Percoll® and 0.2 M to 0.5 M sucrose.  
   
   
       3 . The solution for preserving mammalian early embryos or ES cells by vitrification according to  claim 1  comprising 700 to 900 mg/100 ml NaCl, 15 to 25 mg/100 ml KCl, 8 to 15 mg/100 ml CaCl 2 , 15 to 25 mg/100 ml KH 2 PO 4 , 7.5 to 1.25 mg/100 ml MgCl 2 .6H 2 O, 100 to 130 mg/100 ml Na 2 BPO 4 , 3 to 4 mg/100 ml Na pyruvate, 75 to 125 mg/100 ml glucose, 5 to 10 mg/100 ml antibiotics, 250 to 350 mg/100 ml BSA, and 10% to 15% (v/v) propylene glycol.  
   
   
       4 . The solution for preserving mammalian early embryos or ES cells by vitrification according to  claim 2  comprising 700 to 900 mg/100 ml NaCl, 15 to 25 mg/100 ml KCl, 8 to 15 mg/100 ml CaCl 2 , 15 to 25 mg/100 ml KH 2 PO 4 , 7.5 to 1.25 mg/100 ml MgCl 2 .6H 2 O, 100 to 130 mg/100 ml Na 2 HPO 4 , 3 to 4 mg/100 ml Na pyruvate, 75 to 125 mg/100 ml glucose, 5 to 10 mg/100 ml antibiotics, 250 to 350 mg/100 ml BSA, 10% to 15% (v/v) propylene glycol, 25% to 35% (v/v) ethylene glycol, 15% to 25% (v/v) Percoll®, and 0.2 M to 0.5 M sucrose.  
   
   
       5 . A solution for preserving mammalian early embryos or ES cells by vitrification comprising a solution consisting of 700 to 900 mg/100 ml NaCl, 15 to 25 mg/100 ml KCl, 8 to 15 mg/100 ml CaCl 2 , 15 to 25 mg/100 ml KH 2 PO 4 , 7.5 to 1.25 mg/100 ml MgCl 2 .6H 2 O, 100 to 130 mg/100 ml Na 2 HPO 4 , 3 to 4 mg/100 ml Na pyruvate, 75 to 125 mg/100 ml glucose, 5 to 10 mg/100 ml antibiotics, and 250 to 350 mg/100 ml BSA, with the solution for preservation by vitrification according to  claim 4  dissolved therein at a concentration of 75% to 100% (v/v).  
   
   
       6 . The solution for preservation by vitrification according to any of  claims 1  to  5 , wherein the mammalian early embryos are mammalian 2_cell-stage embryos.  
   
   
       7 . The solution for preservation by vitrification according to  claim 6 , wherein the mammalian 2_cell-stage embryos are rat 2_cell-stage embryos and the mammalian ES cells are primate ES cells.  
   
   
       8 . A reagent kit for preserving mammalian early embryos or ES cells by vitrification comprising (1) a first solution for preserving mammalian early embryos or ES cells by vitrification selected from the group consisting of (a) a solution comprising, as a base material, a phosphate buffer that exclusively contains 10% to 15% (v/v) propylene glycol as polyhydric alcohol, and (b) a solution comprising 700 to 900 mg/100 ml NaCl, 15 to 25 mg/100 ml KCl, 8 to 15 mg/100 ml CaCl 2 , 15 to 25 mg/100 ml KH 2 PO 4 , 7.5 to 12.5 mg/100 ml MgCl 2 .6H 2 O, 100 to 130 mg/100 ml Na 2 PO 4 , 3 to 4 mg/100 ml Na pyruvate, 75 to 125 mg/100 ml glucose, 5 to 10 mg/100 ml antibiotics, 250 to 350 mg/100 ml BSA, and 10% to 15% (v/v) propylene glycol, and (2) a second solution for preserving mammalian early embryos or ES cells by vitrification selected from the roup consisting of (a) a solution comprising, as a base material, a phosphate buffer that exclusively contains 10% to 15% (v/v) propylene glycol and 25% to 35% (v/v) ethylene glycol as polyhydric alcohols and further contains 15% to 25% (v/v) Percoll® and 0.2 M to 0.5 M sucrose, (b) a solution comprising 700 to 900 mg/100 ml NaCl, 15 to 25 mg/100 ml KCl, 8 to 15 mg/10 ml CaCl 2 , 15 mg/100 ml KH 2 PO 4 , 7.5 to 12.5 mg/100 ml MgCl 2 .6H 2 O, 100 to 130 mg/100 ml Na 2 HPO 4 , 3 to 4 mg/100 ml Na pyruvate, 75 to 125 mg/100 ml glucose, 5 to 10 mg/100 ml antibiotics, 250 to 350 mg/100 ml BSA, 10% to 15% (v/v) propylene glycol, 25% to 35% (v/v) ethylene glycol, 15% to 25% (v/v) Percoll®, and 0.2 M to 0.5 M sucrose, and (c) a solution consisting of 700 to 900 mg/100 ml NaCl, 15 to 25 mg/100 ml KCl, 8 to 15 mg/100 ml CaCl 2 , 15 to 25 mg/100 ml KH 2 PO 4 , 7.5 to 12.5 mg/100 ml MgCl 2 .6H 2 O, 100 to 130 mg/100 ml Na 2 HPO 4 , 3 to 4 mg/100 ml Na pyruvate, 75 to 125 mg/100 ml glucose, 5 to 10 mg/100 ml antibiotics, and 250 to 350 mg/100 ml BSA, with the solution for preservation by vitrification according to  claim 4  dissolved therein at a concentration of 75% to 100% (v/v).  
   
   
       9 . The reagent kit for preserving mammalian early embryos or ES cells by vitrification according to  claim 8 , which further comprises a cell-thawing solution for the mammalian early embryos or ES cells preserved by vitrification containing, as a base material, a phosphate buffer containing 0.2 M to 0.5 M sucrose.  
   
   
       10 . The reagent kit for preserving mammalian early embryos or ES cells by vitrification according to  claim 9 , which comprises a cell-thawing solution for the mammalian early embryos or ES cells preserved by vitrification containing, as a base material, a phosphate buffer consisting of 700 to 900 mg/100 ml NaCl, 15 to 25 mg/100 ml KCl, 8 to 15 mg/100 ml CaCl 2 , 15 to 25 mg/100 ml KH 2 PO 4 , 7.5 to 12.5 mg/100 ml MgCl 2 .6H 2 O, 100 to 130 mg/100 ml Na 2 HPO 4 , 3 to 4 mg/100 ml Na pyruvate, 75 to 125 mg/100 ml glucose, 5 to 10 mg/100 ml antibiotics, 250 to 350 mg/100 ml BSA, and 0.2 M to 0.5 M sucrose.  
   
   
       11 . The reagent kit for preservation by vitrification according to any of  claims 8  to  10 , wherein the mammalian early embryos are mammalian 2_cell-stage embryos.  
   
   
       12 . The reagent kit for preservation by vitrification according to  claim 11 , wherein the mammalian 2_cell-stage embryos are rat 2_cell-stage embryos and the mammalian ES cells are primate ES cells.  
   
   
       13 . A method of preserving mammalian early embryos or ES cells by vitrification comprising steps of pretreating mammalian early embryos or ES cells in a first solution for preservation by vitrification selected from the group consisting of (a) a solution comprising, as a base material, a phosphate buffer that exclusively contains 10% to 15% (v/v) propylene glycol as polyhydric alcohol, and (b) a solution comprising 700 to 900 mg/100 ml NaCl, 15 to 25 mg/100 ml KCl, 8 to 15 mg/100 ml CaCl, 15 to 25 mg/100 ml KH 2 PO 4 , 7.5 to 12.5 mg/100 ml MgCl 2 .6H 2 O, 100 to 130 mg/100 ml Na 2 HPO 4 , 3 to 4 mg/100 ml Na pyruvate, 75 to 125 m/100 ml glucose, 5 to 10 mg/100 ml antibiotics, 250 to 350 mg/100 ml BSA, and 10% to 15% (v/v) propylene glycol, and cryopreserving the cells in a second solution for preservation by vitrification selected from the group consisting of (a) a solution comprising, as a base material, a phosphate buffer that exclusively contains 10% to 15% (v/v) propylene glycol and 25% to 35% (v/v) ethylene glycol as polyhydric alcohols and further contains 15% to 25% (v/v) Percoll® and 0.2 M to 0.5 M sucrose, (b) a solution comprising 700 to 900 mg/100 ml NaCl. 15 to 25 mg/100 ml KCl, 8 to 15 mg/100 ml CaCl 2 , 15 to 25 mg/100 ml KH 2 PO 4 , 7.5 to 12.5 mg/100 ml MgCl 2 .6H 2 O, 100 to 130 mg/100 ml Na 2 HPO 4 , 3 to 4 mg/100 ml Na pvruvate. 75 to 125 mg/100 ml glucose, 5 to 10 mg/100 ml antibiotics, 250 to 350 mg/100 ml BSA. 10% to 15% (v/v) 1)ropylene glycol, 25% to 35% (v/v) ethylene glycol, 15% to 25% (v/v) Percoll®, and 0.2 M to 0.5 M sucrose, and (c) a solution consisting of 700 to 900 mg/100 ml NaCl, 15 to 25 mg/100 ml KCl, 8 to 15 mg/100 ml CaCl 2 , 15 to 25 mg/100 ml KH 2 PO 4 , 7.5 to 12.5 mg/100 ml MgCl 2 .6H 2 O, 100 to 130 mg/100 ml Na 2 HPO 4 , 3 to 4 mg/100 ml Na pyruvate, 75 to 125 mg/100 ml glucose, 5 to 10 mg/100 ml antibiotics, and 250 to 350 mg/100 ml BSA, with the solution for preservation by vitrification according to  claim 4  dissolved therein at a concentration of 75% to 100% (v/v).  
   
   
       14 . The method of preserving mammalian early embryos or ES cells by vitrification according to  claim 13 , wherein mammalian early embryos or ES cells are immersed in the first solution for preservation by vitrification at room temperature for 5 to 10 minutes, the first solution is then allowed to stand at 0° C. to 5° C. for 30 seconds to 2 minutes, and the resulting solution is cryopreserved in 40 to 60 times its volume of the second solution for preservation by vitrification.  
   
   
       15 . The method of preserving mammalian early embryos or ES cells by vitrification according to  claim 13  or  14 , wherein the mammalian early embryos are mammalian 2_cell-stage embryos.  
   
   
       16 . The method of preserving mammalian early embryos or ES cells by vitrification according to  claim 15 , wherein the mammalian 2_cell-stage embryos are rat 2_cell-stage embryos and the mammalian ES cells are primate ES cells.